How to Use Reversed-Phase HPLC Chromatography Column

In the column joints at both ends, a stainless steel filter (or screen) is placed at each end of the column tube to block the column packing from being washed out of the column by the mobile phase and lost. The components of the empty column are made of 316# stainless steel, which can withstand the effects of common solvents. However, since chloride-containing solvents are corrosive to a certain extent, pay attention to the use of chloride-containing solvents in the column and connecting pipes for a long time to avoid corrosion.

The separation of the HPLC chromatographic column is carried out between the filler and the mobile phase, and the classification of the liquid chromatographic column is based on the type of filler.
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Use of HPLC Column

1. First flush 10 times the HPLC column volume with n-hexane-acetonitrile (99:1) at a flow rate of 0.5ml/min, and then use chlorine, isopropanol, methanol, methanol-water (50: 50) Crush the pillar;
2. Wash the column with 30 times the column volume of a sodium hydroxide solution with pH 11.0 at a flow rate of 0.5ml/min (note that the pH value must not exceed 11.0), and immediately use water (at a flow rate of 0.5ml/min, 30 times) Column volume) rinse, and then change to mobile phase;
3. When preparing the mobile phase, each component should be measured separately, the smaller proportion should be accurately measured, and the pH value should be adjusted to 0.1;
4. When using in reversed phase conditions, pay special attention to the control of the pH range. The lower the pH, the more the risk of hydrolysis. The higher the proportion of water in the mobile phase, the greater the risk of hydrolysis. The most ideal pH range is between pH3.0-7.0;
5. If the mobile phase to be used also contains buffer salts, it is recommended to use the same proportion of the mobile phase without buffer salts for transition before using the analytical mobile phase to avoid the precipitation of buffer salts in the analytical column.

First of all, let’s talk about whether the HPLC column can be backflushed. Can the chromatographic column be backflushed? If the sieve plates at both ends of the chromatographic column have the same aperture, there will be no problem with backflushing. From the perspective of packing loss, the particle size is 5um. And the aperture of the sieve plate is 2um.

In this case, the forward and recoil will not cause the loss of the packing; in addition, from the perspective of column packing, the HPLC column is packed in the opposite direction to the column arrow, and the direction of the liquid flow path during backflushing is the same, and it is no problem to flush the column with displacement fluid at such a high pressure (usually greater than 40MPa) when the column is installed.

Is it possible that a dozen MPa at a flow rate of 1ml/min will cause the column bed to loosen? What’s more, when backflushing is usually 90% water, or 90% or pure organic phase, what kind of damage can such a low pressure cause to the column bed?

Also, the C18 long chain of the packing is bonded to The free stretch of the carbon chain on the bare silica gel is the most beneficial for the analysis. It does not mean that if you use the carbon chain in one direction for a long time, it will be poured in one direction like water plants. After washing with methanol or acetonitrile, it will return to the free stretch state. Even if they do fall in one direction, wouldn’t it be better to backflush them to restore their original state? So I think backflushing will not affect the bed.